Histology and
Histopathology
Histol Histopathol (2010) 25: 177-187
DOI: 10.14670/HH-25.177
http://www.hh.um.es
Cellular and Molecular Biology
Expression and distribution of the intermediate
filament protein nestin and other stem cell related
molecules in the human olfactory epithelium
Amir Minovi1, Martin Witt2,3, Andreas Prescher4,
Volker Gudziol2, Stefan Dazert1, Hanns Hatt5 and Heike Benecke5
1Department
2Smell
of Otorhinolaryngology, Head- and Neck Surgery, Ruhr-University Bochum, St. Elisabeth Hospital, Bochum, Germany,
and Taste Clinic, Department of Otorhinolaryngology, University of Dresden, Medical School, Dresden, Germany, 3Department
of Anatomy, University of Rostock, Rostock, Germany, 4Department of Molecular and Cellular Anatomy, RWTH Aachen, Germany
and 5Department of Cell Physiology, Ruhr-University Bochum, Bochum, Germany
Summary. The olfactory epithelium (OE) is unique in
regenerating throughout life and thus is an attractive
target for examining neurogenesis. The nestin protein
was shown to be expressed in the OE of rodents and is
suggested to be essentially involved in the process of
regeneration. Here we report the expression and
distribution of nestin in the human OE at RNA and
protein level. Moreover, we analysed the expression
profiles in dependence on age and olfactory capacity.
After sinus surgery, biopsies were taken from the
olfactory epithelium of 16 patients aged 20-80 years
with documented differences in their olfactory function.
Our studies revealed that nestin is constantly detectable
in the apical protuberances of sustentacular cells within
the human OE of healthy adults. Its expression is not
dependent on age, but rather appears to be related to the
olfactory function, as a comparison with specimens
obtained from patients suffering either from persistent
anosmia or hyposmia suggests. Particularly, in the
course of dystrophy, often accompanied with impaired
olfaction, nestin expression was occasionally decreased.
Contrarily, the expression of the p75-NGFR protein, a
marker for human OE basal cells, was not altered,
indicating that at least in the tested samples olfactory
impairment is not connected with abnormalities at the
basal cell level. These observations emphasize an
essential role of nestin for the process of regeneration,
and also highlight this factor as a candidate marker for
sustentacular cells in the human olfactory epithelium.
Offprint requests to: Amir Minovi, MD, Department of
Otorhinolaryngology, Head-and Neck Surgery, Ruhr-University Bochum,
St. Elisabeth Hospital, Bleichstr. 15, 44787 Bochum, Germany. e-mail:
[email protected]
Key words: Human olfactory epithelium, Nestin,
Sustentacular cells, Immunohistochemistry
Introduction
The olfactory epithelium (OE) exhibits a unique
feature among mammalian neural systems, retaining the
ability to regenerate throughout adult life. The procedure
of neurogenesis has been extensively studied in vivo and
in vitro in rodents, as well as in other species (Graziadei
and Graziadei, 1979; Calof et al., 1998; Mackay-Sim
and Chuahb, 2000; Schwob, 2002).
The olfactory epithelium is a pseudostratified
structure consisting of three major cell types, the
olfactory sensory neurons (OSNs), the non-neuronal
sustentacular cells in the apical part of the epithelium,
and the proliferating basal cells located adjacent to the
basal membrane (Graziadei, 1971). Studies on rodent
OE have shown that the latter undergo continuous
mitotic activity and contribute to regeneration, achieved
by differentiation into either neuronal or non-neuronal
cells (Mackay-Sim and Chuahb, 2000; Jang et al., 2003;
Carter et al., 2004; Chen et al., 2004). The perikarya of
newly born receptor neurons then migrate towards the
middle layer of the OE, profoundly changing their
morphology within distinct differential stages (Morrison
and Costanzo, 1992).
In order to understand the mechanisms underlying
development and regeneration numerous studies have
been performed, extensively characterizing the
individual cell types within the rodent OE in terms of
molecular and cellular composition. However, due to
difficult access only a limited number of studies have so
far investigated the histomorphology of the human OE
178
Nestin expression in the human OE
(Arnold et al., 2001; Bianco et al., 2004; Hahn et al.,
2005). Several techniques of OE biopsy have been
developed to obtain intact pieces of epithelia without
permanent damage to the donor site (Lanza et al., 1994;
Feron et al., 1998; Jafek et al., 2002; Lane et al., 2002).
Prior studies have already noted some differences in
immunoreactivity and structure between the rodent and
the human OE (Hahn et al., 2005), indicating that
neurogenesis might differ between both species. To
better understand differences concerning processes,
including birth and differentiation of neural cells in
either species, the necessity for identifying cell type
specific markers substantially increases.
In rodents, at least two populations of basal cells
have been morphologically defined which reside in the
olfactory progenitor layer: the globose basal cells
(GBCs) and the horizontal basal cells (HBCs) (Andres,
1965; Graziadei and Graziadei, 1979). Both can be
distinguished by specific markers, GBC-1 for GBCs
(Goldstein and Schwob, 1996) and cytokeratin for HBCs
(Calof and Chikaraishi, 1989; Yamagishi et al., 1989),
although definite support for either of them as the
ultimate olfactory stem cell is controversial. Contrary to
rodents, in the human OE a morphological
discrimination among basal cells is not possible (Hahn et
al., 2005). However, the level distribution of p75-NGFR
suggests that in humans just one common progenitor
exists. Moreover, these cells are hardly distinguishable
in shape from other cell types in the residual part of the
epithelium.
For immature and mature OSNs molecular markers
such as GAP43 (immature) and the olfactory marker
protein (OMP) have been established (Margolis, 1982;
Baker et al., 1989) that are useful for both species. In
humans, maturating OSNs are dispersed throughout the
middle part of the OE; in rodents, however, there is a
columnar organization, with immature neurons more
basal and mature OMP-positive neurons more apically
oriented.
For sustentacular cells SUS-1 and SUS-4 have been
described to be useful antibodies (Hempstead and
Morgan, 1983, Goldstein and Schwob, 1996). Recently,
nestin immunoreactivity has been detected in
sustentacular cells in the OE of adult rats (Doyle et al.,
2001). Nestin belongs to a highly diverse family of
cytoskeletal factors and forms the VI class of
intermediate filament proteins (Lendahl et al., 1990). For
a long time the protein was assumed to exclusively mark
neuronal progenitors of the central nervous system.
Doyle and coworkers described its significant expression
in sustentacular endfeet which are intimately associated
with the basal lamina. Interestingly, nestin
immunoreactivity was upregulated postbulbectomy and
redistributed to the more apical part of sustentacular
cells e.g. the cell bodies (Doyle et al., 2001). Thus, as a
hypothesis, nestin is involved in cellular reorganization
after damage and/or physiological replacement. This
prompted us to elucidate the expression and distribution
of nestin at the protein and mRNA level within the
human OE. Thereby we intended to highlight similarities
and differences compared to the rodent OE.
Furthermore, we analyzed the expression pattern of
nestin and some other markers depending on the age and
the olfactory capacity of different donors. In addition to
psychophysical examinations the application of
immunohistochemistry-based investigations is
increasingly discussed as a potential diagnostic tool for
olfactory disorders, as well as their peripheric reasons.
Impaired olfaction (e.g. anosmia, hyposmia) is a
prevalent phenomenon (Landis and Hummel, 2006)
often caused by sinonasal diseases (Damm et al., 2004).
For the understanding and treatment of such diseases a
profound knowledge of the basic characteristics of the
human OE in terms of molecular and cellular
composition is essential.
Materials and methods
The study was performed according to the
Declaration of Helsinki on Biomedical Research
Involving Human Subjects. It was approved by the Ethic
Committee of the Ruhr University of Bochum (register
number 2918). Patient data including all relevant
information are listed in Table 1.
Biopsy of OE
OE biopsies were obtained with informed consent
from subjects during sinus surgery or septoplasty.
Subjects with major medical illnesses, including
psychiatric illnesses and metabolic diseases (diabetes,
renal insufficiency) were excluded. Patients with
polyposis were also excluded. 16 subjects, aged 20-80
Table 1. Patient characteristics.
Patient-ID Age Gender Diagnosis Olfactory condition Site of biopsy
1
2
3
4
5
6
7
8
9
10
11
12
13 *
14 *
15
16
17
18
80
65
35
50
70
80
80
21
20
20
52
65
78
48
65
41
48
M
M
M
M
F
M
M
M
F
F
F
M
M
M
M
M
M
M
Post mortem
Post mortem
SD
SD
IA, SD
SD, CRS
CRS
CRS
SD
IA, SD
IA, SD
IA, CRS
CRS
CRS
CRS
CRS
SD
SD
unknown
unknown
Hyposmia
Normosmia
Anosmia
Normosmia
Hyposmia
Hyposmia
Normosmia
Anosmia
Anosmia
Anosmia
Hyposmia
Hyposmia
Hyposmia
Hyposmia
Anosmia
Hyposmia
Regio olfactoria
Regio olfactoria
NS, MT
NS, MT
NS, MT
NS, MT
MT
MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
NS, MT
SD: Septal deviation, IA: idiopathic anosmia, CRS: chronic
rhinosinusitis, NS: Nasal septum; MT: Middle turbinate; *: patients with
Parkinson’s disease.
179
Nestin expression in the human OE
years, participated in OE biopsy. For morphological and
molecular biology purposes, up to four biopsies were
taken from each patient including the area of the lateral
superior wall of the nasal cavity, close to the radix of the
medial turbinate and the opposite dorsal septum, as
described before (Leopold et al., 2000). No biopsies
were obtained from the nasal cleft immediately below
the lamina cribrosa. From most subjects three samples
were immediately fixed in 4% formalin (for
immunohistochemistry), and one sample was immersed
into RNAlater solution (QIAgen, Hilden, Germany) for
mRNA analysis.
Psychophysical studies
All subjects were evaluated concerning their
olfactory capacity by means of a standardized
psychophysical test using “Sniffin’ Sticks”. Olfactory
deficits were assessed by investigating the ability for
odor identification, discrimination and detectionthreshold sensitivity according to Hummel et al. (2007).
Preparation of post-mortem OE tissues
Post-mortem OE tissues were obtained from two
subjects by the Department of Anatomy at the University
of Aachen, Germany. The olfactory function of both
subjects, aged 65 and 80 years, was unknown. At
autopsy, the OE, bony septae, and contiguous cribriform
plate were removed en bloc and fixed for 24 to 36 hours
in 10% neutral buffered formalin. Subsequently, the
samples were decalcified for three weeks in distilled
water containing sodium ethylene-diamine-tetra-acetic
acid, sodium hydroxide, and glycerol at pH 7.1–7.4.
Tissue blocks were then cut into coronal blocks,
dehydrated in graded ethanol solution and xylene, and
embedded in paraffin (Trojanowski et al., 1991; Smutzer
et al., 1998).
Antibodies
Immunolocalization studies were performed using
monoclonal antibodies against the olfactory marker
protein (OMP, Wako Chemicals, 1:5000), nestin
(Chemicon, 1:50), beta-III-tubulin (Acris, 1:60), and
p75-NGFR (Sigma, 1:500).
Immunohistochemistry
Probes were immediately fixed in 10% formalin and
embedded in paraffin. Sections were cut at a thickness of
4 µm. Serial 4-µm sections were placed on slides,
deparaffinized, rehydrated, and dried overnight at 37°C
as previously described (Witt et al., 2009).
Immunohistochemistry was performed using the
peroxidase-antiperoxidase method in combination with
3-3’-diaminobenzidine (DAB). For standard DAB
detection, endogenous peroxidase activity was quenched
using 3% H2O2 diluted in distilled water. Subsequent to
blocking in 1x Tris-buffered saline (TBS) containing
10% appropriate normal serum, sections were incubated
overnight at room temperature with the primary antibody
diluted in TBS containing 1% of normal serum. For
secondary antibody reactions, the sections were washed
in phosphate-buffered saline (PBS) and treated with
either rabbit-anti-goat or goat anti-mouse biotinylated
IgG antibodies (Dako, Copenhagen, Denmark, diluted
1:400 in PBS) for one hour at room temperature.
Primary antibodies were then detected using a
streptavidin-biotin-peroxidase kit (ZytoChem HRP Kit,
Zytomed Systems GmbH, Germany) and visualized by
DAB reaction. Performing sequential double
immunostaining, binding of the first antibody was
visualized by a brown reaction product generated using
the DAB chromogen solution. To detect the second
primary antibody, the AEC (3-amino 9-ethylcarbazole)
chromogen (ZytoChem HRP Kit, Zytomed Systems
GmbH, Germany) was used, yielding a red product. For
better visualization of the nuclei some sections were
counterstained with haematoxylin. The following
controls were performed: (1) omission of the primary
antibody in order to rule out nonspecific binding of the
secondary antibody; and (2) in-parallel stainings of
tissues (e.g., mouse and rat as well as normal human
olfactory epithelium) previously reported to be
immunopositive for the markers tested (Heilmann et al.,
2000; Weiler and Benali, 2005). The summary of all
immunohistochemical analyses is shown in Table 2.
Staining intensities were rated according to the visual
appearance in comparison to the slides and are indicated
by “-” (no staining = 0), “+” (weak staining = 1), “++”
(strong staining = 2), or “+++” (strongest staining = 3).
Depending on the olfactory function two groups were
built: group 1 consists of norm- and hyposmic patients
and group 2 of anosmic patients with no olfactory
function. The results were then compared using the chisquare (χ2) test.
Table 2. Results of Immunohistochemistry.
Patient-ID Olfactory condition
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Unknown
Unknown
Hyposmia
Normosmia
Anosmia
Normosmia
Hyposmia
Hyposmia
Normosmia
Anosmia
Anosmia
Anosmia
Hyposmia
Hyposmia
Hyposmia
Hyposmia
Anosmia
Hyposmia
p75-NGFR
++
++
++
+++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
beta-III-T
+++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
+
++
Nestin OMP
+++
+++
++
+++
+
+
+
++
+++
+
+
++
++
+
++
+
+
+++
+++
++
+
+
+++
++
+++
+++
+
+
+
++
++
++
++
+
++
180
Nestin expression in the human OE
RT-PCR analysis
For reverse transcription-PCR (RT-PCR), total RNA
was isolated from olfactory mucosa (RNeasy Mini Kit,
Qiagen, Hilden, Germany). The quality of the prepared
RNA samples was assessed by A260/A280 measurements
and agarose gel electrophoresis. Equal amounts of RNA
were reverse-transcribed using reverse transcriptase
(Fermentas RevertAid cDNA synthesis kit; Fermentas
GmbH, St.Leon-Roth, Germany) with oligo-p(dT)15
primers and random hexamer primers following the
manufacturer’s protocol in a final volume of 20 µl. The
PCR was performed by using Taq-polymerase
(Ampliqon, Copenhagen, Denmark) and gene-specific
primers synthesized according to published cDNA
sequences of the olfactory marker protein (OMP), nestin,
p75-NGFR, and beta-III-tubulin. As an endogenous
control we used GAPDH, a housekeeping gene that is
highly expressed in human tissues.
Reactions were run on a PCR Cycler (Techne TC
512, Burlington, NJ, USA) under the following cycling
conditions: 94°C for 2 min followed by 35 cycles of
amplification. Each cycle included 30 s denaturation at
95°C, 30 s annealing at 62°C (except for b-III-tubulin:
58.5°C) and 30 s synthesis at 72°C. Amplified PCRproducts were visualized after electrophoresis in 1.5%
agarose gel containing ethidium bromide. The specificity
of the PCR amplificates was confirmed by DNA
sequencing.
Results
From a total of 42 biopsies taken from the
presumptive olfactory area, 27 specimens (64%)
contained olfactory epithelium. In all other biopsies
regular or metaplastic respiratory epithelium was
observed independent of the age and the olfactory
condition. Immunohistochemical analysis was
performed with OE biopsies of sixteen patients with ages
ranging from 20-80 years. Specimens of a control group
of healthy persons were compared with patients
suffering either from persistent anosmia or hyposmia.
One healthy subject was also subjected to RT-PCR
analysis together with seven other patient samples to
validate gene expression on mRNA level.
In the post-mortem nasal sample from patient 1, OE
was mainly detectable in the region of the superior
turbinate and in the insertion of the middle turbinate.
The largest continuous area containing OMP-positive
cells had an extension of about 1.5 mm. In the second
post-mortem sample (patient 2), functional olfactory
mucosa with OMP-positive cells was observed in a small
area of the middle turbinate. All other regions were
covered with regular or dysplastic respiratory
epithelium. In all biopsies olfactory sensory neurons
were visualized by antibody-staining of the olfactory
marker protein. As shown in Figure 1, age had no
obvious impact on the morphology of the epithelia or on
the number of OMP-positive cells, as a comparison
between the OEs derived from a 20, a 50 and an 80 year
old subject revealed (Fig. 1a-c). In all cases sensory
neurons were equally distributed among the middle part
of the epithelium. Thus, densities of OMP-positive cells
in normosmic patients are comparable between young
and older subjects. The OSN area is apically restricted
by a mono cell layer of sustentacular cells, exhibiting a
typical shape, and by the basal cell layer adjacent to the
lamina propria, both devoid of staining. The transition to
the respiratory epithelium (RE), indicated by an arrow in
Figure 1c, is characterized by an abrupt lack of OMPpositive cells.
Contrary to these observations, a comparison
between OMP-staining of the epithelia taken from
patients with olfactory impairment with that of a healthy
Fig. 1. Human olfactory
epithelium (hOE) obtained
from three patients with
normosmia at the age from 20
(a, patient 9), 50 (b, patient 4)
and 80 years (c, patient 6).
Olfactory sensory neurons
(OSNs) were stained with an
OMP-antibody using the DAB
procedure. In (b) and (c) cells
were additionally
counterstained by
haematoxylin. The arrow in
(a) indicates an olfactory
nerve bundle. The arrow in (c)
denotes the transition from
olfactory to respiratory
epithelium. SUS:
sustentacular cells; OSN:
olfactory sensory neurons;
BC: basal cells. Scale bars: a,
50 µm; b, c, 20 µm.
Nestin expression in the human OE
181
person indicated that the amount of OSNs is dependent
on the olfactory function (Fig. 2). While the epithelia of
normosmic patients exhibited a high density of equally
distributed OSNs (Fig. 2a), the number of OMP-positive
cells was obviously decimated in that of anosmic
persons (Fig. 2c).
For patients with designated hyposmia no strongly
visible reduction in the number of OSNs could be
observed (Fig. 2b). Application of the chi-square (χ2)
test revealed a significant reduction of OMP-positive
cells in the OE of anosmics compared with hyp- and
normosmics (Chi-square=13.2, df=2, p=0.001). For
anosmics, these findings were partially accompanied by
mild signs of dystrophia, which is expressed as a
reduction in thickness of the epithelium, as well as by
increasingly indistinct cell contours. However, most but
not all OE biopsies from anosmic individuals strongly
tended to a profound degeneration, as indicated by
dysplastic morphology of the epithelium.
We next examined the presence and distribution of
nestin in the human OE and its dependence on different
olfactory functions. First, we concentrated on the
expression profile of nestin in the OE of healthy
subjects. The presence and localization of the protein
was examined by immunostaining with a specific nestin
antibody. Figure 3 shows the representative results of
nestin staining in the olfactory epithelium of a
normosmic subject. The protein was clearly detectable
by persistent staining of the apical region of the OE
where sustentacular cells are typically located. More
precisely, counterstaining by haematoxylin illustrates
that the microvillar fringe of the sustentacular cells is the
location of major nestin expression (Fig 3a-c). In
contrast, the bodies of the sustentacular cells showed no
staining. Only poor signals were obtained in the basal
cell layer where the proliferating neuronal progenitors
reside. According to the observations described by
Doyle and coworkers faint antibody reactions in this area
might derive from staining of sustentacular endfeet
(Doyle et al., 2001). The part of the OE where the OMP-
Fig. 2. OMP-staining of human OE biopsies derived from patients with
different olfactory function. OSNs were stained with an anti-OMP
antibody. The epithelium of a normsomic subject (a, patient 4) shows a
high density of equally distributed OMP-positive OSNs. In comparison,
in a hyposmic subject (b, patient 3) the number of OSNs is slightly
reduced and the OE shows mild dystrophia. For an anosmic subject (c,
patient 12) noticeable degeneration of the OE accompanied by a low
density of OSNs was observed. Scale bars: 20 µm.
Fig. 3. Expression and distribution of the nestin protein in the human OE
(patient 4). a. Nestin (DAB) is predominantly expressed in the apical
processes of sustentacular cells of the OE, but absent in the respiratory
epithelium (RE). The arrow indicates the transition between both
epithelia. (Counterstaining with haematoxylin). b-c. Higher magnification
of nestin-stained OE and RE (100x). Nestin expression appears in the
apical part of the OE, while in the basal cell layer just weakly stained
spots can be observed. Scale bars: 20 µm.
182
Nestin expression in the human OE
positive OSNs are located is exempt from nestin
expression. Figure 4a shows the spatial separation from
OSNs by parallel staining of nestin and OMP, in
combination with a DAB and an AEC reaction,
respectively. As similarly detectable for OMP-staining,
the presence of nestin-positive cells abruptly interrupts
at the transition to the respiratory epithelium, which is
indicated by an arrow in the corresponding figure (Fig.
3a-c).
To further analyze the dependence of nestin
expression on olfactory function, sections of the OE
Fig. 4. Expression of nestin (DAB, brown) and OMP (AEC, red) protein
in the OE of a normosmic (a, patient 9), hyposmic (b, patient 7) and an
anosmic subject (c, patient 12). Nestin is clearly expressed in the
sustentacular cells of the normosmic subjects and slightly reduced in the
OE of hyposmic patients. In the case of anosmic patients protein
expression varied between noticable reduction to complete lack of
staining. SUS: sustentacular cells; OSN: olfactory sensory neurons; BC:
basal cells. Scale bars: 20 µm.
from normosmic, hyposmic and anosmic subjects were
taken and compared concerning their staining pattern
obtained by the antibody reaction (Fig. 4). Nestin
immunoreactivity, which is clearly present in the
sustentacular cell layer of normosmic patients, was
slightly reduced in the OE of subjects suffering from
hyposmia (cf Fig. 4a,b). A comparison of the expression
patterns among eight hyposmic subjects revealed that
Fig. 5. Expression of p75-NGFR (DAB, brown) in the OE of normosmic
(patient 4) (a), hyposmic (patient 3) (b), and anosmic (patient 11) (c)
patients. In either case p75-NGFR-staining could be detected in the
basal cells independent of the olfactory function. Counterstaining with
haematoxylin. Scale bars: 20 µm.
Nestin expression in the human OE
staining intensities varied to some extent (cf Table 2),
which also might be dependent on the prior location
within the OE. In contrast, in the OE of anosmics, nestin
expression was found to be noticeably reduced; at least
in one specimen tested staining completely failed (Table
2, patient 10). However, due to subject dependent
variations, differences between groups just missed the
level of significance (Chi-square=5.67, df=2, p=0.059).
Clearly visible contours of all cell types demonstrate that
reduced staining was not due to a lack of the
sustentacular cell layer which might have occurred at
biopsy preparation. Due to strong interindividual
183
differences concerning the morphology of the human
OE, more patient samples will have to be analyzed to
obtain definite evidence for the dependence of nestin
expression on the olfactory function.
Degeneration of the OE, which often emerges as a
reduction of thickness, and of OSN quantities might be a
consequence of loss of basal cells or at least of impaired
basal cell function. Therefore, we tried to identify
differences at basal cell level within the OE of
hyposmics and anosmics in comparison to that of
normosmic subjects. For this reason we performed
comparative immunostaining analysis on the epithelia of
Fig. 6. Expression of beta-III-T
in the OE of a hyposmic
patient (Nr. 18): b-III-T
antibody (a) labels not only
mature OSN and its dendritic
and axonal processes, but
also non-mature OSN, as well
as non-olfactory neurons, here
probably trigeminal nerve
fibers (arrowheads). b.
lesioned olfactory epithelium
with still intact OMP reactive
cells (b, arrows). Scale bar:
100 µm.
Fig. 7. a. RT-PCR amplification of
OMP mRNA in normosmic (lane 1),
hyposmic (lanes 2-5), and anosmic
(lane 6-8) patients. Most of them
showed a positive band for OMP gene
products, except patient 4. Subject 5
shows only a very weak band
(compared to overexpressed GAPDH
signal). Even normosmic individuals
presented relatively weak OMP bands.
b. This survey illustrates the
occurrence of mRNAs for several gene
products associated with neurogenetic
processes. mRNAs for p75-NGFR,
nestin, and beta-III-tubulin are
expressed in all subjects investigated.
184
Nestin expression in the human OE
Table 3. RT-PCR analysis of patients with different olfactory capacities.
Patient-ID Olfactory condition GAPDH p75-NGFR beta-III-T nestin OMP
5*
6 **
13
14
15
16
17
18
Anosmia
Normosmia
Hyposmia
Hyposmia
Hyposmia
Hyposmia
Anosmia
Hyposmia
+++
+++
++
+++
+++
+++
+++
+++
++
+
nd
+
++
++
++
++
++
+
nd
++
++
++
++
++
+
+
nd
+
+
+
+
+
+
+
+
+
+
++
++
*: same patient as ID 5 in Table 2; **: same patient as ID 6 in Table 2
normosmics and of hyp- and anosmics with visible
degeneration by using the p75-NGFR antibody. p75NGFR was previously described as a marker of neuronal
precursor cells in the human OE (Hahn et al., 2005).
Interestingly, comparable intensities of p75-NGFR could
be observed in all cases tested (Fig. 5). Thus, the basal
cell layer seems not to be affected, arguing against a loss
of basal cells as the reason for impaired olfaction in
these patient cases. Concerning the expression of BetaIII-Tubulin (b-III-T) we could not see any difference
between the groups. As shown in Figure 6a b-III-T
expression was seen not only in mature OSN and its
dendritic and axonal processes, but also in non-mature
OSN, as well as in non-olfactory neurons. In parallel to
the above described investigations, we performed RTPCR analysis on patient samples, summarized in Table
3. Figure 7a shows the RT-PCR amplificates of OMP
and the GAPDH housekeeping genes in total RNA
extracted from the nasal mucosa of 8 subjects. We
observed OMP bands of variable, although not
quantified, intensities in all subjects, regardless of their
olfactory capacity. Only one hyposmic individual did not
show any amplificate for OMP-mRNA. Figure 7b
compiles the results of gene products for the p75-NGF
receptor (occurring in basal cells), beta-III-tubulin, and
nestin. We observed all of these products in similar
intensities.
Discussion
Impaired olfaction strongly influences quality of life;
about 5% of the general population suffers from
anosmia, which is characterized by complete loss of
olfactory function (Hummel et al., 2007). Besides that,
abnormalities like impaired olfaction, also described as
e.g. partial anosmia or hyposmia, appear with high
occurrence. Although the reasons for these diagnostic
findings can be manifold and therefore often remain
unknown, they mainly reveal a sinonasal nature
(Hummel and Huttenbrink, 2005). To enhance our
understanding of the particular molecular mechanisms of
olfactory diseases it is essential to investigate the
epithelial characteristics of certain patients with specific
olfactory function.
Olfactory impairment at an older age is most likely
due to a decreased secretion of the nasal mucus, and thus
to a diminished mucociliary transport (Rawson, 2006).
Studies that describe minimal age-related changes in the
rodent OE at molecular, light microscopic and
ultrastructural levels support the assumption that agerelated loss in olfactory function is not an intrinsic
feature of the aging process (Hinds and McNelly, 1981;
Apfelbach and Weiler, 1991; Naguro and Iwashita,
1992). In contrast to that, for human beings increasing
age significantly decreases the probability of obtaining
olfactory tissue in biopsy material (Paik et al., 1992). At
an age of about 60 years the OE becomes more
frequently interspersed with patches of respiratory
epithelium and is extensively displaced in elderly
subjects. That is in line with our observations with
detectable amounts of olfactory tissue in only 27 out of
42 samples. As regards this, differences between rodents
and humans are probably based on the fact that these
studies were performed with animals, which were not
really old compared to the age humans reach.
Although we did no quantification, our results
suggest that age has no influence on the number of
OSNs, as a comparison of OMP-positive cells in the OE
of young and elderly subjects revealed. Mature OMPpositive OSNs were similarly distributed among the
major part of the OE. This is in contrast to findings from
Paik et al. (1992), who reported decreased numbers of
OSNs in elderly individuals. Contrary results might be
explained by differences between autopsy material and
surgical biopsies. On the other hand our data is in good
agreement with a study of Feron et al. (1998), who did
not observe age-related differences comparing various
patient OEs.
Since techniques of OE biopsies were successfully
established, several studies concentrated on the
histological and histopathological examination of the
human olfactory epithelium derived from subjects
suffering from diseases associated with olfactory
impairment, e.g. neurodegenerative diseases like
schizophrenia or Parkinson’s disease (Trojanowski et al.,
1991; Crino et al., 1995; Arnold et al., 1998, 2001;
Smutzer et al., 1998; Duda et al., 1999; Feron et al.,
1999). More recent studies (Lee et al., 2000) analyzed
the OE of anosmic patients with chronic sinusitis. In
concert with their findings, we also observed a
significant decrease of olfactory sensory neurons in the
OE of all anosmic subjects tested. The identity of OSNs
was proven by consistent detection of the olfactory
marker protein by antibody staining. However, more
quantitative assessment e.g. by RealTime-PCR was not
performed as the aforementioned studies clearly
demonstrated analogous findings. In morphological
aspects, the orderly arrangement characteristic of the
healthy OE was widely lost in most anosmics,
demonstrating a degenerative appearance. In contrast, an
obvious enhancement of OMP-positive cells in
normosmics over hyposmics could not always be
observed. To confirm these observations further analysis
Nestin expression in the human OE
of olfactory epithelia of additional hyposmic donors is
indispensable. Kern (2000) showed signs of
inflammation in some patients with chronic sinusitis.
They mainly observed an infiltration of the OE with
lymphozytes. In our patient biopsies we did not detect
any inflammation of the olfactory mucosa as indicated
by HE-staining of the basal lamina.
The major aim of our study was the analysis of the
general expression pattern and the spatial distribution of
the intermediate filament protein nestin within the
human OE at protein and mRNA level. Nestin is known
to be expressed by various cell types during
development. However, its expression is usually
transient and does not persist into adulthood, except for
neural precursors of the central nervous system
(Hockfield and McKay, 1985; Frederiksen et al., 1988;
Lendahl et al., 1990; Fishell et al., 1993). Upon
differentiation nestin is down-regulated and displaced by
tissue-specific intermediate filament proteins (Lendahl et
al., 1990; Sejersen and Lendahl, 1993). Thus, for a long
time the protein was considered to be one of the best
markers for neural progenitors in mammals (Lendahl et
al., 1990; Chiasson et al., 1999; Rao, 1999). In 2001,
Doyle and co-workers described the interesting finding
that nestin is also expressed in the OE of adult rats.
However, its expression generally was detected in the
endfeet of sustentacular cells (SUS), which are located
within the basal cell layer where neural precursors
reside. More interestingly, after bulbectomy, when
regenerative events probably get accelerated, nestinpositive staining significantly appeared in the apical part
of the OE, where typically sustentacular cell bodies are
located (Doyle et al., 2001). These findings strongly
support an essential participation of nestin in
regenerative processes of the rodent OE. The exact role
of SUS cells has not been established in human OE so
far. In rodents SUS cells have been thought to regulate
the ionic composition of the mucus layer and to detoxify
chemical olfactory stimuli (Getchell et al., 1984; Chen et
al., 1992). In contrast to OSNs the population of SUS
cells is not lost after unilateral olfactory bulbectomy.
Exposing the OE to methyl bromide gas (MeBr) leads to
an elimination of greater than 95% of SUS cells and
OSNs, whereas a large portion of the basal cells is
spared (Schwob et al., 1995; Schwob, 2002). Following
this, a rapid regeneration of the SUS cells and OSNs is
observed within weeks. It is presumed that SUS cells can
be generated by globose basal cells of the rodent OE
(Schwob, 2002).
For the human OE, RT-PCR analysis of olfactory
mucosa already provided evidence for the expression of
nestin at RNA level (Murrell et al., 2005). In concert
with that, our immunohistochemical results clearly show
that nestin is also expressed at protein level in the
healthy human adult OE; nestin could be detected in all
normosmic patients. However, the vast nestin-staining in
the human OE is rather found in the typical microvillar
protuberances of sustentacular cells than in the area of
basal cells. At the basal lamina almost no staining was
185
visible, most probably co-localizing with the endfeet of
sustentacular cells. This hypothesis is based on findings
from the aforementioned publication by Doyle and coworkers, where co-staining with the specific SUS-4
antibody uncovered these structures as the endfeet
processes of sustentacular cells in the adult rat OE
(Doyle et al., 2001).
Doyle and colleagues hypothesized that in rodents
nestin might play a role in the migration of newborn
olfactory neurons on the scaffolding of sustentacular
cells, essentially taking part in the regeneration of the
OE. Based on this assumption, we suggest that the
human OE exhibits a generally more active state of
regeneration than occurs in healthy rodents. Permanently
generated OSN may then need guidance to reach their
appropriate place in the epithelium, which could find
support by a complex network of intermediate filament
proteins such as nestin. This accompanies a similar
hypothesis that was raised in the course of comparative
expression analysis of p75-NGFR in the rodent and
human OE (Hahn et al., 2005). Here, p75-NGFR
revealed to be a suitable marker for proliferating basal
cells in the human adult OE, which in turn was quite
surprising due to the fact that in the rodent OE p75NGFR is exclusively expressed during embryogenesis
but not in adulthood (Vickland et al., 1991).
Additionally, our observation of comparable p75-NGFR
expression in the human adult OE of various aged
donors is in good agreement with data provided by Hahn
and colleagues (Hahn et al., 2005).
Contrary to the situation in rodents, nestin might be
a suitable marker of sustentacular cells in the human OE,
which is further supported by the lack of age-related
differences in expression profiles in the OE of
normosmic patients. In contrast, our results of
comparative analysis of biopsy material taken from
healthy persons with that derived from subjects with
impaired olfaction (e.g. hyposmia, anosmia) suggest a
dependence of the nestin expression on the olfactory
function. Whereas for all hyposmics only weak
reduction in nestin-expression was visible, antibody
staining was more obviously reduced in the OE of the
most anosmic donors; in one anosmic patient there was a
complete loss of staining.
In some cases the OE of anosmic subjects showed
strong degeneration, which was indicated by a dramatic
decrease in thickness, accompanied by indistinct cell
contours. It is unclear whether the detection of nestin
was negatively affected here due to a lack of
sustentacular cells. At least in the OE of some anosmics
reduction of nestin staining cannot be attributed to cell
degeneration as indicated by typical shapes of
sustentacular cells. Unlike in rodents the human OE
shows great intra- and interindividual varieties
depending not only on the diagnosis but also on the site
of biopsy. Therefore our results concerning the functiondependent nestin expression have to be interpreted
critically. Taken together, the present study highlights
new differences between the rodent and human olfactory
186
Nestin expression in the human OE
epithelium. Here we demonstrate permanent expression
of the nestin protein in the apical part of sustentacular
cells of the adult human olfactory epithelium, which is in
contrast to rats. Moreover, the presented results provide
a first indication for the dependency of the expression
profiles of nestin mRNA and protein on the olfactory
capacity.
Acknowledgements. We are greatful to Susane Kannabey and Kerstin
Pehlke for excellent technical assistance and to Günter Gisselmann and
Thomas Hummel for helpful discussion on the manuscript. This study
was supported by the grant FoRUM (Nr. F596-07) from the Ruhr
University Bochum.
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Accepted August 18, 2009